1. Field of the Invention
The invention relates to a protein from plasma membranes of adipocytes which has specific binding affinity to phosphoinositolglycans.
2. Description of the Background
The role of phospholipids and phospholipases in trans-membrane signaling is firmly established. Equally well-established is the concept of anchoring proteins into cell membranes through a covalently linked glycosylphosphatidylinositol (GPI), and the precise chemical structure of the GPI anchor has been worked out for several GPI-anchored proteins, such as acetylcholinesterase (AchE) from human erythrocytes, rat Thy-1, and several coat proteins of parasites like the variant surface glycoprotein (VSG) from Trypanosoma brucei. Lipid anchoring occurs through phosphatidylinositol (PI), which consists of a diacyl- or an alkylacyl glycerol type phospholipid. Since the latter occurs, among others, in mammalian anchors, and differs from the bulk PI present in membranes, it could provide a novel molecular species involved in the generation of second messengers derived from GPIs. Signaling by GPIs is of special interest as these lipid-anchored molecules do not span the membrane, but in most cases are embedded in the outer half of the lipid bilayer. The signal-mediated release from the cell membrane of GPIs has been demonstrated for a variety of endocrine and paracrine molecules, ranging from hormones to growth factors. The involvement of GPIs in transmembrane signaling and their intracellular effects seems by now established, but little is known about the signaling pathway leading to the observed metabolic effects.
The notion that GPI-anchored molecules possess signaling properties results from early experiments in which it was shown that the binding of insulin to its receptor activates the hydrolysis of GPIs. A low-molecular-weight substance was identified that mimics certain actions of insulin on metabolic enzymes. This substance has an inositol glycan structure and is produced by the insulin-sensitive hydrolysis of a GPI in the plasma membrane. Although the GPI precursor for the inositol glycan enzyme modulator was originally thought to be structurally analogous to the GPI membrane protein anchor, there are distinct differences in the carbohydrate moiety between the signal transducing GPI and the GPI anchor of membrane proteins. The GPI-membrane protein anchor invariably consists of the trimannose core followed by an ethanolamine phosphate, which provides the link to the C-terminal amino acid of the attached protein.
Regulated GPI hydrolysis is not only restricted to insulin but has been observed with a number of other hormones.
In practically all cases, the stimulation of cells by hormones or growth factors leads to a transient release of GPI-anchored proteins from the cell surface. Most of the receptors for these agonists are either tyrosine kinase receptors or receptors coupled to tyrosine kinases.
Many of the proteins involved in insulin action have been identified at the molecular level. The insulin receptor is a transmembrane tyrosine kinase, which when activated by insulin binding, undergoes rapid autophosphorylation and phosphorylates a number of intracellular substrates, among them one or more 50-60 kDa proteins, including the Shc, a 15 kDa fatty acid binding protein and several so-called insulin receptor substrate proteins, IRS-1/2/3/4. After tyrosine phosphorylation, the IRS polypeptides act as docking proteins for several Src homology 2 domain-containing adaptor molecules and enzymes, including phosphatidylinositol 3-kinase (PI 3-K), Grb2, SHP2, Nck, and Fyn. The interaction between the IRS proteins and PI 3-K occurs through the p85 regulatory subunit of the enzyme and results in an increase in catalytic activity of the p110 subunit. PI 3-K is essential for many insulin-sensitive metabolic processes, including stimulation of glucose transport and glycogen synthesis. In all cases in which there is stimulation of tyrosine phosphorylation of IRS proteins, there is concomitant docking of these proteins to the p85 subunit of PI 3-K and, with the exception of the cross-talk between the insulin and angiotensin signaling systems, this docking was associated with stimulation of PI 3-K activity.
In addition to the identification of the signal-transduction pathways leading directly from the insulin receptor to down-stream targets, several cross-talks have been delineated between signaling transmission by insulin and other hormones/growth factors or diverse exogenous stimuli, which either mimic (to a certain degree) or modulate in a positive or negative fashion metabolic and/or mitogenic insulin action in various cellular systems. Since none of these ligands activates the insulin receptor kinase directly, their signaling pathways may converge with that of insulin at a more distal signaling step. This property is shared by phosphoinositolglycan-peptide (PIG-P) molecules of different type as for example for PIG-P prepared from the glycosylphosphatidylinositol anchor of yeast Gce1p which mimic metabolic insulin action to a significant degree without concomitant induction of insulin receptor kinase activity.
Positive cross-talk of phosphoinositolglycans (PIG) and PIG-peptides (PIG-P) to the insulin signal transduction cascade in insulin-responsive target cells involves redistribution of glycosylphosphatidylinositol (GPI)-anchored plasma membrane proteins (GPI protein) and dually acylated non-receptor tyrosine kinases from detergent-resistant glycolipid-enriched plasma membrane raft domains of high cholesterol content (hcDIGs) to rafts of lower cholesterol content (IcDIGs).
In isolated rat adipocytes the primary target of PIG-P is localized in hcDIGs. Radiolabeled PIG-P, Tyr-Cys-Asn-NH—(CH2)2—O—PO(OH)O-6Manα1-2)2Manα1-6Manα1-4GluN1-6Ino-1,2-(cyclic)-phosphate (YCN-PIG) as well as radiolabeled and lipolytically cleaved GPI protein (IcGce1p) from Saccharomyces cerevisiae, from which YCN-PIG has been derived, bind to hcDIGs in saturable fashion but not to IcDIGs, microsomes or total plasma membranes. Binding of both YCN-PIG and IcGce1 is specific, as it is completely abolished either by excess of chemically synthesized unlabeled YCN-PIG or by pretreatment of the adipocytes with trypsin and subsequent NaCl or N-ethylmaleimide (NEM) indicating that YCN-PIG is recognized by a cell surface receptor. Binding of PIG-P is considerably increased in hcDIGs from adipocytes pretreated with GPI-specific phospholipases C compatible with lipolytic removal of endogenous ligands, such as GPI proteins/lipids. Binding affinity is highest for YCN-PIG, followed by the combination of the separate constituents, Tyr-Cys-Asn-NH—(CH2)2—OH(YCN) plus HO—PO(H)O-6Manα1 (Manα1-2)-2-Manα1-6Manα1-4GluN1-6Ino-1,2-(cyclic)-phosphate (PIG37), and the peptide variant, YMN-PIG. PIG37 and YCN alone exhibit intermediate and low affinity. Incubation of adipocytes with YCN-PIG diminishes subsequent labeling by [14C]NEM of the 115 kDa polypeptide released from the cell surface by sequential trypsin/NaCl-treatment. These data show that in rat adipocytes insulin-mimetic PIG(-P) are recognized by a trypsin/NaCl/NEM-sensitive 115 kDa protein of hcDIGs which acts as receptor for GPI proteins.
Several types of DIGs seem to exist in the same cell. Caveolae represent special DIGs in terminally differentiated cells which form flask-shaped invaginations driven by the abundant expression of the marker and structural protein, caveolin 1-3.
Caveolae which account for 20% of the plasma membrane surface area in adipocytes participate in receptor-mediated potocytosis, endocytosis, transcytosis and signal transduction. In isolated rat adipocytes IcDIGs of low cholesterol/caveolin content exhibiting high buoyant density (according to sucrose density gradient centrifugation) can be discriminated from typical hcDIGs with high cholesterol/caveolin content characterized by low buoyant density. The major fraction of GPI proteins, such as Gce1 and Nuc, as well as of dually acylated proteins, such as the NRTK Non Receptor Tyrosine Kinase, pp59Lyn, are located at hcDIGs. In response to insulin-mimetic stimuli such as synthetic PIG or the sulfonylurea, glimepiride, both GPI proteins and NRTKs are translocated from hcDIGs to IcDIGs. This redistribution is not caused by loss of their lipid modification.
The polar core glycan head group without (PIG) or with (PIG-P) adjacent amino acids from the carboxyl-terminus of the GPI protein polypeptide moiety provides the molecular basis of the distribution of GPI proteins between hcDIGs and IcDIGs in the basal state and their redistribution in response to insulin-mimetic stimuli.
GPI proteins are cell surface antigens, ectoenzymes, receptors or cell adhesion molecules expressed in eucaryotes from yeast to man and anchored to the outer leaflet of the plasma membrane by a covalently attached glycosylphosphatidylinositol (GPI) lipid moiety. Despite the lack of a transmembrane domain, they have been implicated in signal transduction across the plasma membrane.
The finding that GPI proteins associate with specialized lipid raft domains, so-called detergent-insoluble glycolipid-enriched rafts, DIGs, rather than with distinct transmembrane binding/linker proteins demonstrates the possibility of lipid-lipid interactions as the major coupling mechanism for signal transduction mediated by GPI proteins.
The basic structural element of DIGs is a lateral assembly of (glyco)sphingolipids and cholesterol which adopts a liquid-ordered (Io) organization distinct from that of adjacent liquid-disordered (Id) regions in the membrane lipid bilayer. The plasma membranes of mammalian cells contain cholesterol (30-50 mol %) and a mixture of lipids with preference for the Id domains (e.g. phosphatidylcholines with unsaturated tails) and lipids bearing saturated acyl chains with preference for Io domains (e.g. [glyco]sphingolipids and GPI lipids). Cholesterol is thought to contribute to the tight packing of lipids in Io domains by filling interstitial spaces between lipid molecules, and the formation of Io domains is seen only within certain ranges of cholesterol concentration.
Insulin is a very important hormone, which exerts a significant effect on the metabolism of the body. In the general terms it promotes anabolic processes and inhibits catabolic processes. Specifically it increases the rate of synthesis of glycogen, fatty acids and protein, and inhibits the breakdown of protein and glycogen. A vital action of the hormone is to stimulate cells from a liver, muscle and fat to remove glucose, some other sugars and amino acids from the blood.
Bovine insulin consists of two polypeptide chains, polypeptide A containing 21 AA and polypeptide B containing 30 AA, which are joined by two —S—S— (disulfide bridges). This same structural pattern occurs in insulin of many mammals including humans.
The structure is compact cylinder-like with only the carboxyl end of the B chain sticking out from the rest of the protein. There are many hydrophobic residues, which interact to form a central hydrophobic core, and interdispersed are some polar residues on either side that further stabilize the protein. Three disulfide bridges clamp the structure together, two inter-chain and one intra-chain.
A common feature in the biosynthesis of many proteins, but in particular for proteins exported from cells, is that the protein is produced in a precursor form then modified to produce the final form during storage and before release. Insulin is synthesized by a group of cells in the pancreas called Islets of Langerhans, stored in granules then released into the blood when required.
When insulin is first synthesized it consists of a 100 AA single polypeptide chain consisting of a signal sequence of 16 AA, a B chain, a C chain called connecting chain of 33 AA, and a A chain. This structure is called pre-proinsulin (PPI). It is thought that the signal region is responsible for directing the PPI from the site of synthesis to the ER (endoplasmic reticulum) in the cell, which collect and package the insulin to form storage granules. When located in the ER, the signal peptide is removed by a protease enzyme.
Diabetes mellitus is a chronic disease that requires long-term medical attention both to limit the development of its devastating complications and to manage them when they do occur. Diabetes is associated with acute and chronic complications as hypoglycemia, diabetic ketoacidosis and hyperosmolar non-ketotic syndrome.
Type 1 diabetes generally occurs in young, lean patients and is characterized by the marked inability of the pancreas to secrete insulin because of autoimmune destruction of the beta cells. The distinguishing characteristics of a patient with type 1 diabetes is that if insulin is withdrawn, ketosis and eventually ketoacidosis develop. These patients are, therefore, dependent on exogenous insulin to sustain their lives.
Type 2 diabetes typically occurs in individuals older than 40 years who have a family history of diabetes. Type 2 diabetes is characterized by peripheral insulin resistance with an insulin-secretory defect that varies in severity. These defects lead to increased hepatic gluconeogenesis, which produces fasting hyperglycemia. Most patients (90%) who develop type 2 diabetes are obese, and obesity itself is associated with insulin resistance, which worsens the diabetic state.
A variety of other types of diabetes, previously called “secondary diabetes”, are caused by other illnesses or medications. Depending on the primary process involved (i.e., destruction of pancreatic beta cells or development of peripheral insulin resistance), these types of diabetes behave similarly to type 1 or type 2 diabetes. The most common are diseases of the pancreas that destroy the pancreatic beta cells (e.g., hemochromatosis, pancreatitis, cystic fibrosis, pancreatic cancer), hormonal syndromes that interfere with insulin secretion (e.g., pheochromocytoma) or cause peripheral insulin resistance (e.g., acromegaly, Cushing syndrome, pheochromocytoma), and drug-induced diabetes (e.g., phenytoin, glucocorticoids, estrogens).
Diabetes mellitus is characterized by inappropriate regulation of serum glucose levels. In Type 1 diabetes an autoimmune attack on the endocrine pancreas results in progressive and irreversible destruction of the insulin secreting beta cells. Loss of insulin action on insulin-sensitive target cell glucose uptake and metabolism results. Type 2 diabetes has several etiologies, most often reflected in cellular resistance to insulin action, also with attendant alterations in the regulation of serum glucose levels. Insulin acts through a disulfide-bonded heterotetrameric cell surface receptor comprised of an extracellular alpha subunit coupled via disulfide bonds to a transmembrane and intracellular beta subunit. In Type 1 diabetes, absence of the ligand with normal cellular receptor structure and function is most often the cause of the subsequent metabolic defects. Hormone replacement therapy in the form of daily insulin injections supplies the ligand for receptor action, though not necessarily in a normal physiologic fashion. In Type 2 diabetes, resistance to the action of insulin often underlies the disease with some of the resistance due to defects in receptor action.
It is known in case of insulin resistance that a higher amount of insulin is required to set on the insulin signaling cascade by the insulin receptor. The present invention is related to a cell membrane protein of adipocytes which is able to stimulate glucose uptake by circumventing the insulin receptor triggered signaling pathway. This provides for a powerful solution of the problem not to have in hands a screening tool to identify compounds which could act as alternatives for insulin.